Bone marrow stem cell compositions, methods of making, and uses thereof

ABSTRACT

Provided herein are bone marrow stem cell compositions and implants and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of currently pending U.S. Utilityapplication Ser. No. 15/591,678, filed May 10, 2017, which is acontinuation-in-part of currently pending U.S. Utility application Ser.No. 15/525,418, filed May 9, 2017, which is the 35 U.S.C. § 371 nationalstage application of PCT Application No. PCT/US2016/056617, filed onOct. 12, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/240,318, filed on Oct. 12, 2015, and U.S. ProvisionalPatent Application No. 62/240,348, filed on Oct. 12, 2015.

BACKGROUND

Bone and tissue grafting are surgical procedures that replace missingbone or other tissues to repair bone and other joint or other tissuedamage. Generally, bone has the ability to regenerate completely buttypically need some sort of scaffold to do so. Current bone grafts canbe natural (allograft or autologous) or synthetic compositions that havesimilar mechanical properties as bone (e.g. hydroxyapatite). Despite theregenerative nature of bone, conditions are not always ideal for healingof the bone graft. Indeed, despite the efficacy of modern internalfixation techniques, infection, poor vascularity, malnutrition, andsubstantial bone or soft tissue loss can impede effective osteogenesis.As such there exists a need for improved compositions and methods forincreasing the healing efficacy of existing bone and tissue graftingtechniques.

SUMMARY

Provided herein are methods of making a composition that can containliving stems cells derived from bone marrow, that can contain the stepsof harvesting bone marrow from a donor to obtain harvested bone marrow,where the bone marrow contains bone marrow cells, selectively lysing orrinsing the bone marrow cells to remove undesired cells (e.g. red andwhite blood cells) but retaining viable stem cells (e.g. bone marrowmesenchymal stem cells), decontaminating the remaining viable stemcells, and cryopreserving the stem cells remaining. The method canfurther include the step of wherein an amount of the composition thatcan contain viable stem cells is implanted in or otherwise administeredto a subject in need thereof.

Also provided herein are compositions and implants that can containviable stem cells, where the compositions can be made by a method thatcan include the steps of harvesting bone marrow from a donor to obtainharvested bone marrow, where the bone marrow contains bone marrow cells,selectively lysing or rinsing the bone marrow cells to remove undesiredcells (e.g. red and white blood cells) but retaining viable stem cells(e.g. bone marrow mesenchymal stem cells), decontaminating the remainingviable stem cells, and cryopreserving the stem cells. The viable stemcells can contain one or more bioactive factors, which can be a proteinselected from the group of: epidermal growth factor, an insulin-likegrowth factor, a fibroblast growth factor, vascular endothelial growthfactor, osteoprotegerin, and osteopontin. The bioactive factors can bepresent in the composition or implant at a concentration of about 0 μg/gto about 100 mg/g. The bioactive factor can be bone morphogeneticprotein 2 and can be present at a concentration of at least 1 pg/g. Thebioactive factor can be acidic fibroblast growth factor and can bepresent at a concentration of at least 1 pg/g. The bioactive factor canbe basic fibroblast growth factor and can be present at a concentrationof at least 1 pg/g. The bioactive factor can be vascular endothelialgrowth factor and can be present at a concentration of at least 1 pg/g.

Also provided herein are methods of treating a subject in need thereofthat can include the step of implanting or administering a compositionor an implant to the subject in need thereof, where the composition orimplant can include viable stem cells, where the composition is made bya method that can include the steps of harvesting bone marrow from adonor to obtain harvested bone marrow, where the bone marrow containsbone marrow cells, selectively lysing or rinsing the bone marrow cellsto remove undesired cells (e.g. red and white blood cells) but retainingviable stem cells (e.g. bone marrow mesenchymal stem cells),decontaminating the remaining viable stem cells, and cryopreserving thestem cells. The subject in need thereof can have a bone fracture. Thesubject in need thereof can need a bone fusion. The subject in needthereof can be in need of a spinal fusion.

Also provided herein are implants that can contain an amount of a bonescaffold and a composition that can contain viable stem cells derivedfrom bone marrow, where the composition further can contain an acid at aconcentration or amount that retains stem cell viability. The scaffoldcan be in the form of a block/structures, chips, or morsels. The viablestem cells can contain a bioactive factor, which can be present in thecomposition at a concentration of at least at least 1 pg/g. Thebioactive factor can be present in the composition at a concentration ofabout 0 μg/g to about 100 mg/g. The bioactive factor can be selectedfrom the following group of: an epidermal growth factor, an insulin-likegrowth factor, a fibroblast growth factor, vascular endothelial growthfactor, osteoprotegerin, and osteopontin, and combinations thereof. Thebioactive factor can be insulin like growth factor-1. The bioactivefactor can be α-fibroblast growth factor and can be present at aconcentration of at least 1 pg/g. The bioactive factor can beβ-fibroblast growth factor. The β-fibroblast growth factor can bepresent at a concentration of at least 1 pg/g. The bioactive factor canbe vascular endothelial growth factor. The vascular endothelial growthfactor can be present at a concentration of at least 1 pg/g.

Also provided herein are methods that can contain the step of implantingin or administering to a subject in need thereof an implant that cancontain an amount of a composition or implant that can contain viablestem cells derived from bone marrow, which can contain a bioactivefactor. The composition or implant can further contain an acid at aconcentration or amount that retains the viability of the stem cells.The subject in need thereof can be in need of a bone graft or a bonefusion.

Also provided herein are methods that can include the step of implantingor administering an implant that can contain an amount of a scaffold anda composition or implant that can contain viable stem cells derived frombone marrow. The composition or implant can further contain an acidpresent at an amount or concentration that retains the viability of thestem cells, where the subject in need thereof can have a bone fracture,diseased bone, joint fracture, a diseased joint, or a combinationthereof.

Also provided herein are methods of fusing a portion of a spine, wherethe method can contain the step of implanting or administering animplant that can contain an amount of a scaffold and composition thatcan contain viable stem cells derived from bone marrow. The compositionor implant can further contain an acid at an amount or concentrationthat retains viability of the stem cells.

Also provided herein are methods of grafting bone where the method cancontain the step of implanting or administering an implant that cancontain an amount of a scaffold and a composition that can contain anamount of viable stem cells that are derived from bone marrow. Thecomposition can further contain an acid at an amount or concentrationthat can retain the viability of the stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows an embodiment of a method of making a bone marrow stem cellcomposition or implant.

FIG. 2 shows an embodiment of a method of making a bone marrow stem cellcomposition or implant.

FIG. 3 shows an embodiment of a method of making a bone marrow stem cellcomposition or implant.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited unless expressly stated otherwise. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.Further, the dates of publication provided could be different from theactual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, biochemistry, botany and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

Definitions

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within ±10% of the indicated value, whichever is greater.

As used herein, “adipocyte” refers to a cell type also known as alipocyte or fat cell. Adipocytes are the cells that primarily composeadipose tissue, specialized in storing energy as fat.

As used herein, “additive effect” refers to an effect arising betweentwo or more molecules, compounds, substances, factors, or compositionsthat is equal to or the same as the sum of their individual effects.

As used herein, “administering” refers to an administration that isoral, topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra-joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques.

As used herein, “allogeneic” refers to involving, derived from, or beingindividuals of the same species that are sufficiently geneticallydifferent so as to interact with one another antigenically.

As used herein, “allograft” refers to a graft that is derived from onemember of a species and grafted in a genetically dissimilar member ofthe same species.

As used herein, “autograft” refers to a graft that is derived from asubject and grafted into the same subject from which the graft wasderived.

As used herein, “autologous” refers to being derived from the samesubject that is the recipient.

As used herein, “bioactive” refers to the ability or characteristic of amaterial, compound, molecule, or other particle that interacts with orcauses an effect on any cell, tissue and/or other biological pathway ina subject.

As used herein, “bioactive factor” refers to a compound, molecule, orother particle that interacts with or causes an effect on any cell,tissue, and/or other biological pathway in a subject.

As used herein, “biocompatible” or “biocompatibility” refers to theability of a material to be used by a patient without eliciting anadverse or otherwise inappropriate host response in the patient to thematerial or a derivative thereof, such as a metabolite, as compared tothe host response in a normal or control patient.

As used herein, “cell,” “cell line,” and “cell culture” include progeny.It is also understood that all progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included.

As used herein, “complete extracellular matrix” refers to extracellularmatrix that has all components (proteins, peptides, proteoglycans, andthe like) present and may or may not include other cells that areembedded in the extra cellular matrix.

As used herein, “concentrated” used in reference to an amount of amolecule, compound, or composition, including, but not limited to, achemical compound, polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, that indicates that the sample isdistinguishable from its naturally occurring counterpart in that theconcentration or number of molecules per volume is greater than that ofits naturally occurring counterpart.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purposes and included to minimize ordistinguish the effect of variables other than an independent variable.

As used herein, “culturing” refers to maintaining cells under conditionsin which they can proliferate and avoid senescence as a group of cells.“Culturing” can also include conditions in which the cells also oralternatively differentiate.

As used herein, “decellularized extracellular matrix” refers to completeextracellular matrix that has been processed to remove any cellsembedded within the extracellular matrix.

As used herein, “diluted” used in reference to an amount of a molecule,compound, or composition including but not limited to, a chemicalcompound, polynucleotide, peptide, polypeptide, protein, antibody, orfragments thereof, that indicates that the sample is distinguishablefrom its naturally occurring counterpart in that the concentration ornumber of molecules per volume is less than that of its naturallyoccurring counterpart.

As used herein, “donor” refers to a subject from which cells or tissuesare derived. As used herein, “effective amount” can refer to an amountsufficient to effect beneficial or desired results. An effective amountcan be administered in one or more administrations, applications, ordosages.

As used herein, “endogenous” refers to a compound, substance, ormolecule originating from within a subject or donor, including theircells or tissues.

As used herein, “extra cellular matrix” refers to the non-cellularcomponent surrounding cells that provides support functions to the cellincluding structural, biochemical, and biophysical support, includingbut not limited to, providing nutrients, scaffolding for structuralsupport, and sending or responding to biological cues for cellularprocesses such as growth, differentiation, and homeostasis.

As used herein, “extracellular matrix component” refers to a particularcomponent. By way of a non-limiting example, an extracellular matrixcomportment can be a specific class of comments (e.g. proteoglycans) orindividual component (e.g. collagen I) that is separated or isolatedfrom the other extracellular components. These components can be madesynthetically.

As used herein, “exogenous” refers to a compound, substance, or moleculecoming from outside a subject or donor, including their cells andtissues.

As used herein, “filler” refers to a substance used to fill a cavity ordepression. The filler can fill the depression such that it is levelwith the surrounding area or that the cavity is filled, such that thedepth of the depression or volume of the cavity is decreased, or suchthat the area that was the depression is now raised relative to theareas immediately surrounding the depression.

As used herein “hydrogel” refers to a network of hydrophilic polymerchains that are dispersed in water. “Hydrogel” also includes a networkof hydrophilic polymer chains dispersed in water that are found as acolloidal gel.

As use herein, “immunogenic” or “immunogenicity” refers to the abilityof a substance, compound, molecule, and the like (referred to as an“antigen”) to provoke an immune response in a subject.

As used herein, “implant” or “graft,” as used interchangeably herein,refers to cells, tissues, or other compounds, including metals andplastics, that are inserted into the body of a subject.

As used herein, “isolated” means separated from constituents, cellularand otherwise, with which the polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, are normally associated innature. A non-naturally occurring polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, does not require “isolation” todistinguish it from its naturally occurring counterpart.

As used herein, “negative control” refers to a “control” that isdesigned to produce no effect or result, provided that all reagents arefunctioning properly and that the experiment is properly conducted.Other terms that are interchangeable with “negative control” include“sham,” “placebo,” and “mock.”

As used herein, “physiological solution” refers to a solution that isabout isotonic with tissue fluids, blood, or cells.

As used herein, “positive control” refers to a “control” that isdesigned to produce the desired result, provided that all reagents arefunctioning properly and that the experiment is properly conducted.

As used herein, “preventative” refers to hindering or stopping a diseaseor condition before it occurs or while the disease or condition is stillin the sub-clinical phase.

As used herein “scaffold” can refer to bone or processed bone material.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” includes a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term farmanimal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama,alpaca, turkey, and the like.

As used herein, “therapeutic” refers to treating or curing a disease orcondition.

As used herein “self-assembling peptides” refer to peptides whichundergo spontaneous assembly into ordered nanostructures.“Self-assembling peptides” include di-peptides, lego peptides,surfactant peptides, molecular paint or carpet peptides, and cyclicpeptides.

As used herein, “specific binding” refers to binding which occursbetween such paired species as enzyme/substrate, receptor/agonist,antibody/antigen, and lectin/carbohydrate which may be mediated bycovalent or non-covalent interactions or a combination of covalent andnon-covalent interactions. When the interaction of the two speciesproduces a non-covalently bound complex, the binding which occurs istypically electrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, “specific binding” occurs between a pairedspecies where there is interaction between the two which produces abound complex having the characteristics of an antibody/antigen orenzyme/substrate interaction. In particular, the specific binding ischaracterized by the binding of one member of a pair to a particularspecies and to no other species within the family of compounds to whichthe corresponding member of the binding member belongs. Thus, forexample, an antibody preferably binds to a single epitope and to noother epitope within the family of proteins.

As used herein, “syngeneic” refers to subjects or donors that aregenetically similar enough so as to be immunologically compatible toallow for transplantation, grafting, or implantation.

As used herein, “synergistic effect,” “synergism,” or “synergy” refersto an effect arising between two or more molecules, compounds,substances, factors, or compositions that is greater than or differentfrom the sum of their individual effects.

As used herein “xenograft” or “xenogeneic” refers to a substance orgraft that is derived from one member of a species and grafted or usedin a member of a different species.

Discussion

Bone grafting is a common procedure performed for a variety oforthopedic and dental reasons. Many materials have been developed thatcan be used for bone graft procedures. Such materials include, but arenot limited to, autograft, allograft, and synthetic bone graftmaterials. While these materials have enjoyed a certain amount ofclinical success, donor morbidity when using autograft materials,adverse recipient immune response when using allograft materials, andlimited bone remodeling and low osteoconductivity that can be observedwhen using synthetic materials. Attempts to improve the clinicalperformance of all types of materials have employed the use ofrecombinant or synthetic bioactive factors that are involved in thebone-remodeling process. While there have been attempts to obtainbioactive factors directly from various tissue sources, all have reliedupon harsh chemicals or other conditions to isolate the bioactivefactors, which does not retain viable cells and can lead to low yieldsof viable bioactive factors such and reduce clinical performance of thebioactive factors obtained. Further, the variability in the amount andtype of cells or bioactive factors obtained directly from tissue sourcesdue to the methods used to obtain the bioactive factors severely limitsthis approach for any practical clinical purpose.

With the aforementioned shortcomings in mind, described herein arecompositions and implants that can include a composition that cancontain viable stem cells derived from bone marrow provided herein. Thecompositions provided herein can, in some embodiments, overcome one ormore of the shortcomings of existing implant materials. Othercompositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Bone Marrow Stem Cell Compositions and Implants

Bone Marrow Stem Cell Compositions

Bone marrow is the soft, spongey, gelatinous tissue found in the hollowspaces in the interior of bones. Bone marrow contains stem cells thatare supported by a fibrous tissue called the stroma. There are two maintypes of stem cells in bone marrow: (1) hematopoietic stem cells and (2)bone marrow mesenchymal stem cells (bmMSCs). bmMSCs can differentiateinto a variety of cells types including without limitation, fibroblasts,chondrocytes, osteocytes, myotubes, stromal cells, adipocytes,astrocytes, and dermal cells. In addition to bmMSCs, bone marrow stromacontains other types of cells including fibroblasts (reticularconnective tissue) macrophages, adipocytes, osteoblasts, osteoclasts,red blood cells, white blood cells, leukocytes, granulocytes, platelets,and endothelial cells.

The bone marrow stem cell compositions and implants described andprovided herein can contain viable bone marrow mesenchymal stem cells,fibroblasts, chondrocytes, osteocytes, red blood cells, white bloodcells, leukocytes, granulocytes, platelets, and/or osteoclasts. In someembodiments, the bone marrow stem cell compositions and implantsselectively contain bone marrow stem cells (e.g. bone marrow mesenchymalstem cells) and not other types of cells (e.g. immunogenic cells, whiteblood cells, red blood cells, platelets, leukocytes, and/orgranulocytes). In some aspects, the viable bone marrow stem cells cancontain bioactive factors, which can be proteins. The proteins can beintracellular proteins or membrane associated proteins. Such proteinsinclude, transforming growth factors (TGF-β1, TGF-β2), epidermal growthfactor (EGF), hepatocyte growth factor (HGF), insulin-like growthfactors (IGFs) (e.g. IGF-1), fibroblast growth factors (FGFs) (e.g. aFGF(acidic fibroblast growth factor) and bFGF (basic fibroblast growthfactor)), vascular endothelial growth factor (VEGF), platelet derivedgrowth factor-BB (PDGF-BB), osteoprotegerin (OPG), and osteopontin(OPN).

The bone marrow stem cell compositions and implants can contain viablebone marrow stem cells that have been cryopreserved. In someembodiments, the s bone marrow stem cell compositions and implants canbe liquid or flowable solution. In some embodiments, the bone marrowstem cell compositions and implants can be frozen in such a way that theviability of at least some of the bone marrow stem cells is retained.Suitable cryopreservation techniques to retain the viability of bonemarrow stem cells will be appreciated by one of ordinary skill in theart. In some embodiments, bone marrow stem cell compositions andimplants can be frozen to less than about 0° C. such as −10, −20, and−80° C. or more. The bone marrow stem cell compositions and implants donot inherently contain recombinant proteins. The bone marrow stem cellcompositions and implants as described herein can increase theefficiency of implant and/or graft integration and/or healing over thatof the bone marrow stem cells if present in the context of complete bonemarrow or other complete bodily fluid or tissue.

Additionally, the bone marrow stem cell compositions and implantsdescribed herein can lack or contain a reduced amount of the immunogenicproteins, cells, and other components that are present in complete bonemarrow and/or other complete bodily fluid or tissue. The bone marrowstem cell compositions and implants provided herein, in someembodiments, do not include a recombinant or synthetic protein or otherbioactive factor. In other words, in some embodiments the bone marrowstem cell compositions and implants can be non-recombinant bone marrowstem cell compositions and implants. In other embodiments, the bonemarrow stem cell compositions and implants can contain one or morerecombinant factors.

The bone marrow stem cells contained in the bone marrow stem cellcomposition or implants can contain a bioactive factor. Any particularbioactive factor in the bone marrow stem cells can be present in thebone marrow stem cell composition or implant at a concentration of 0μg/g to about 100 mg/g of isolated protein in the final product (e.g.composition or implant), cryopreserved, or otherwise provided. The bonemarrow stem cell compositions and implants can include at least about 1pg/g aFGF, about 1 pg/g to about 100 μg/g aFGF, about 1 ng/g to about100 ng/g aFGF, or about 20 to about 40 ng/g aFGF. The bone marrow stemcell compositions and implants can include at least about 1 pg/g bFGF,about 1 pg/g to about 100 μg/g bFGF, about 1 ng/g to about 100 ng/gbFGF, or about 20 ng/g to about 40 ng/g bFGF. The concentration of VEGFin the bone marrow stem cell compositions and implants can be at leastabout 1 pg/g, or about 1 pg/g to about 100 μg/g VEGF, about 1 ng/g toabout 150 ng/g VEGF, or about 60 ng/g to about 90 ng/g VEGF. The bonemarrow stem cell compositions and implants can include at least 1 pg/gPDGF, or about 1 pg/g PDGF to about 100 pg/g PDGF, about 500 pg/g toabout 500 ng/g PDGF, about 900 pg/g to about 100 ng/g PDGF, or to about950 pg/g to about 50 ng/g PDGF. The bone marrow stem cell compositionsand implants can include at least 1 pg/g OPN, or about 1 pg/g OPN toabout 100 μg/g OPN, about 500 pg/g OPN to about 500 ng/g OPN, about 900pg/g to about 100 ng/g OPN, or to about 950 pg/g to about 50 ng/g OPN.

Additionally, the bone marrow stem cell compositions and implants canalso contain an amount of a suitable acid or water at an amount orconcentration that can retain the viability of the bone marrow stemcells present in the composition or implant. In some embodiments, theacid is a residual or other amount of the acid that can be used toselectively lyse the bone marrow cells to retain viable bone marrow stemcells (e.g. bone marrow mesenchymal stem cells) but lyse other celltypes (e.g. red and white blood cells). In some embodiments, the acidcan be acetic acid. In other embodiments, water can be used toselectively lyse the cells.

In some embodiments, the bone marrow stem cell compositions and implantscomposition can include a stabilizer composition or stabilizercompounds. Suitable stabilization compositions can include, but are notlimited to glycerol, glucose, sucrose, amino acids, preservatives,antibiotics, antivirals, antifungals, pH stabilizers, osmostablizers,anti-inflammants, anti-neoplastics, chemotherapeutics, immunomodulators,chemoattractants, growth factors, anticoagulants, nutrients, orcombinations thereof. The stabilization solution can increase shelf lifeof the bone marrow stem cell compositions and implants and maintain theviability of the bone marrow stem cells present in the bone marrow stemcell compositions and implants prior to use.

In some embodiments, a bone marrow stem cell compositions and implantscomposition can be reconstituted. This can result in a dilution of thebone marrow stem cells within the bone marrow stem cell compositions andimplants. In some embodiments, the bone marrow stem cell compositionsand implants can be concentrated using a suitable concentration methodsuch that the number of viable cells per cc or mg of composition orimplant is increased in comparison to the starting material The bonemarrow stem cell compositions and implants can be diluted/concentratedfrom 0.1 to 100 fold, 0.1 to 50 fold, 0.1 to 20 fold, or 0.1 to 5 fold.Dilution or concentration can occur before or during use.

Implants Including a Bone Marrow Stem Cell Composition

Provided herein are bone marrow stem cell implants that can contain agrafting scaffold materials (also referred to herein as “scaffolds”)that can include a bone marrow stem cell composition provided elsewhereherein that can have one or more viable stem cells of the compositionbound adsorbed, absorbed, or otherwise attached to or associated with ascaffold material. The scaffolds can be bone material.

The bone marrow stem cell compositions can be any bone marrow stem cellcomposition provided herein. The bone marrow stem cell compositionsincluding or not including the scaffold material can be cryopreserved.The bone marrow stem cell compositions and implants, proteins and/orother bioactive factor(s) can become diluted when contacted with bodilyfluids, for example, when the bone material containing the bone marrowstem cell composition are implanted in or otherwise administered to asubject in need thereof. As described elsewhere herein, the bone marrowstem cell compositions can contain an amount of an acid or water in anamount or concentration that can retain the viability of the bone marrowstem cells contained in the composition or implant. The acid can beacetic acid. The acid can be a residual amount left over from the methodof producing the bone marrow stem cell composition.

Scaffold Materials

The scaffold material can be a bone material, such as allograft,autograft, or xenograft bone. The bone material can be wholly orpartially demineralized. The scaffold material can be sterilized.

Methods of Making the Bone Marrow Stem Cell Compositions and Implants

Described herein are methods for producing compositions containingviable bone marrow stem cells. In some embodiments, the viable bonemarrow stem cells can attach to or otherwise associate with a scaffoldprior to delivery to or use in a subject. The soluble proteincompositions prepared by the methods described herein can have a greateramount and/or concentration of bone marrow stem cells and/or additionalbioactive factor(s) contained within the viable stem cells, and/or lessimmunogenicity than other osteoinductive/osteostimulatory compositions,implants, or devices incorporating complete bone marrow and/or othercomplete bodily fluids or tissues. The bone marrow stem cellcompositions and implants can contain bioactive proteins such asacidic-FGF, basic-FGF, IGF, BMP-7, HGF, VEGF, PDGF-BB, OPG, and OPN.

Attention is first directed to FIG. 1, which shows an embodiment of amethod of producing a soluble protein composition from bone marrow. Thebone marrow can be harvested from a cadaver or from a living subject.The method can begin by harvesting bone marrow from a donor 100. Thedonor can be a cadaver or can be a living subject. The bone marrow canbe autologous, allogeneic or xenogenic. The bone marrow can be harvestedin any way generally known in the art. The bone marrow can be obtainedfrom cancellous, corticocancellous, and/or cortical bone. The harvest ofthe bone marrow may also include bone prior to washing. After the bonemarrow has been harvested, the bone marrow can be washed 520 in asolution. The wash solution may contain water, saline, antibiotic,antiseptic, antifungal, or crystalloid solution. In some embodiments,the wash solution is only water. Washing can take place at anytemperature. In some embodiments, washing takes place at least at 20° C.In some embodiments, washing takes place at about 20° C. to about 37° C.In further embodiments, washing takes place at about 20° C. to about 40°C. In some embodiments, the washing takes place at 37° C. Heating thebone marrow during washing can facilitate the reduction in viscosity orremoval of undesired fat (adipocytes) from other types of bone marrowcells. The washing/heating step can be performed under physicalagitation in a shaker incubator. In some embodiments, shaking ca beconducted at about 10-300 rpm for up to about 24 hours. In someembodiments, shaking can be conducted for about 20, 40, 60, 120, 240,260, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23 or 24 hours. When physical agitation is used, the conditions of thephysical agitation are such that the viability of at least some of thebone marrow stem cells present in the bone marrow cells is retained.

During washing and/or heating 520, some of the bone marrow derived cellscan be lysed. Under some conditions, substantially all of the bonemarrow derived cells remain intact and viable after washing and/orheating 520. After heating and/or washing, the bone marrow cells can beselectively lysed 500. In some embodiments, the bone marrow derivedcells can be selectively lysed using a selective lysing solutioncontaining water, salt, or an acid configured to osmotically selectivelyse some cells but not others that are resistant to the lysisconditions. In some embodiments the cells that are resistant to theselective lysing conditions are bone marrow stem cells (e.g. bone marrowmesenchymal stem cells). In some embodiments, the selective lysingsolution is just water. In some embodiments, the washing solution andthe selective lysing solution can be the same solution. In someembodiments, the selective lysing solution is acetic acid. In someembodiments, the selective lysis solution contains 0.5% v/v or lessacetic acid. In some embodiments the lysing solution that contains thebone marrow and/or marrow-rich bone is mixed with pre-heated water. Insome embodiments, the bone marrow or marrow-rich bone is selectivelylysed 500 for about 60 minutes. In other embodiments, the bone marrow ormarrow-rich bone is incubated in the selective lysing solution withshaking.

After selective lysing 500, the lysate that can contain viable cells canbe optionally fractionated via centrifugation 130 to separate outparticles present in the lysate based on their size or density. Suchcentrifugation techniques that can be employed include, but are notlimited to, differential centrifugation, rate-zonal centrifugation, andisopycnic centrifugation. In embodiments where centrifugation is used toseparate particles in the lysate based on density, a suitable densitygradient medium can be used. Suitable density gradient mediums include,but are not limited to, sucrose, glycerol, sorbitol, Ficoll® medium,polysucrose, dextrans, CsCl, Cs₂SO₄, KBr, Diatrizoate, Nycodenz® medium,Histodenz™ medium, iodixanol, Histopaque® mediums, ACCUSPIN® medium, andPercoll® medium. One of ordinary skill in the art will appreciate thatthe type of medium used is dependent on the type of particle(s) that isdesired to be separated out. One or more rounds of centrifugation can beapplied to the lysate to further separate out different particles in thelysate. In some embodiments, the desired fraction contains viable bonemarrow stem cells, such as bone marrow mesenchymal stem cells. In someembodiments, the lysate is centrifuged at about 100 to about 1,000,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, to about20000 rpm for about 1 to about 600 minutes. In some embodiments, thelysate is centrifuged at about 1 to about 4000×g. The lysate, in someaspects, can be centrifuged for about 10 minutes. In some aspects, thelysate can be centrifuged at room temperature or at about 4° C.

After the optional fractionation 130, the desired fraction that cancontain viable bone marrow stem cells can be removed from thecentrifuged lysate. The bone marrow stem cell containing fraction canthen be optionally cryopreserved, otherwise stored, further processed,or used 140 using a suitable technique. With the general processdescribed, attention is directed to FIGS. 2-3. As shown in FIG. 6, themethod where the harvested bone marrow can be selectively lysed 500prior during or prior to lysing (FIG. 5) can optionally include the stepof filtering 300 the obtained fraction that contains the viable bonemarrow stem cells after centrifugation 130. As shown in FIG. 2, afterfractionation by centrifugation 130 the fraction containing the viablebone marrow stem cells can be further filtered using a suitablefiltration technique to remove additional undesired components that canremain in the fraction. Suitable filtration techniques can include, butare not limited to, size exclusion techniques and/or affinitypurification techniques, immunoseparation techniques, and charged basedseparation techniques. In some embodiments, additional undesiredcomponents can include, but are not limited to, nucleic acids such asDNA and RNA, and other compounds such as hemoglobin, globin proteins,cell fragments, cell membrane molecules and other molecules that canstimulate an immune response in a subject. In some embodiments, thefilter can be low protein binding. In some embodiments, the filter canbe high DNA binding.

In some embodiments, the filter can preferentially bind or retain onecell type over another or other components in the retained fraction.Suitable materials for some filters used in the filtration step 300,include, but are not limited to, Teflon® membranes, nylon membranes,PVDF (polyvinylidene) membranes, polypropylene, cellulose acetate, PES(polyethersulfone), regenerated cellulose, glass fiber, and PTFE(polytetrafluorethylene. In some embodiments, the filter can have a sizecutoff of about 0.1 to about 3.0 μM. In some embodiments multiplefilters can be used, such as in a serial filtration system. In such asystem, multiple types of filters can be used. The system can include atleast two filters that differ in material and size cut offs. In someembodiments, polypropylene filters (e.g. size cut offs of 30 μm and 10μm can be used), a glass fiber filter with a size cutoff of about 2.7 μmcan be used, and/or a series of cellulose acetate filters (8 μm, 5 μm, 3μm, 1.2 μm, 0.8 μm, 0.45 μm and final one of 0.2 μm) can be used tofilter. The filters can be configured as syringe filters, disc filters,vacuum filter systems, bottle top vacuum filters, tube top vacuumfilters, or centrifuge tube filters.

After filtering 300, the filtrate that contains the viable bone marrowstem cells can be cyropreserved, stored, further processed, and/or usedas previously described. As shown in FIG. 3, the methods (FIG. 1 andFIG. 2) where the harvested bone marrow can be selectively lysed 500prior to or during lysing can also include the optional step of adding astabilization solution 200 a,b after centrifugation 130 and/orfiltration 300.

It will be appreciated that other steps can be included in any of themethods described herein. In some embodiments, the method can include apH altering step where an acid or a base or an acidic or basic solutioncan be added to product of any step in any method to result in a productthat is acidic (pH less than 7), basic (pH greater than 7), or neutral(pH of 7). In some embodiments, after lysing, the lysate or product fromany other subsequent step can be made more acidic, neutral, or basic asdesired. In embodiments, the bone marrow stem cell compositions andimplants containing bone marrow stem cells (e.g. bone marrow mesenchymalstem cells) contains an acid that was introduced in the lysing step(e.g. 500). In other embodiments, the stabilization solution can containan acid or base that can result in an acidic, basic, or neutralsolution.

In some embodiments, the method can include a concentration step, wherethe product of any step in any embodiment of the method can beconcentrated by a suitable technique. In some embodiments, the methodcan include a dilution step, where the product of any step in anyembodiment of the method can be diluted by a suitable technique.Suitable concentration techniques include but are not limited to,dehydration techniques (described elsewhere herein) and centrifugationbased techniques. Other concentration techniques will be appreciated bythose of skill in the art.

Methods of Making Implants Containing a Bone Marrow Stem CellCompositions

Methods of making the scaffold material, including bone material, aregenerally known in the art. Methods of making the bone marrow stem cellcompositions are described herein. In some embodiments, the scaffoldmaterial can be introduced during the production of making a bone marrowstem cell composition where the scaffold material is mixed in at a step,such as the initial washing and/or selective lysing step with theinitial starting bone marrow material. In other embodiments, the bonemarrow stem cell composition can be mixed with the scaffold materialafter generating the bone marrow stem cell composition or duringadministration of the stem cell composition to a subject.

Methods of Using the Bone Marrow Stem Cell Compositions and Implants

The bone marrow stem cell compositions and implants described hereinthat can contain viable bone marrow stem cells. In some embodiments, thebone marrow stem cell compositions and implants described herein thatcan contain viable bone marrow stem cells can contain an acid at anamount or composition that retains cell viability of the stem cells inthe composition or implant. The bone marrow stem cell compositions andimplants described herein that can contain viable bone marrow stem cellscan be implanted into or otherwise administered to a subject in needthereof. In some embodiments, an effective amount of the bone marrowstem cell compositions and implants described herein can be implanted orotherwise administered to a subject in need thereof. When implanted oradministered, the bone marrow stem cell compositions and implantsdescribed herein can be diluted by the bodily fluids of the subject.

The bone marrow stem cell compositions and implants described herein toa suitable scaffold described elsewhere herein or a device. The scaffoldcan be flowable or non-flowable.

The bone marrow stem cell compositions and implants described herein canbe applied to a scaffold or implant which is already present in asubject or can be implanted into a subject in need thereof. In otherembodiments, the bone marrow stem cell compositions and implantsdescribed herein can be applied directly into a scaffold already presentin the subject in need thereof.

In some embodiments, the method can include the step of implanting orotherwise administering a bone marrow stem cell compositions andimplants described herein to a subject in need thereof. In someembodiments, a method of treating a subject in need thereof can includethe step of implanting or otherwise administering bone marrow stem cellcompositions and implants described herein to the subject in needthereof.

In some embodiments, the subject in need thereof needs a bone graft orbone fusion. In some embodiments, the subject in need thereof has a boneand/or joint fracture or disease. In some embodiments, the subject inneed thereof needs a spinal fusion. In some embodiments the compositionsdescribed herein can be used in patients with low bone density toprophylactically help reduce, delay, or prevent bone loss or fracture.

In some embodiments, a method of fusing a portion of the spine, wherethe method includes the step of implanting or administering bone marrowstem cell compositions and implants described herein described herein toa subject in need thereof. In some embodiments, a method of bonegrafting, where the method includes the step of implanting oradministering bone marrow stem cell compositions and implants describedherein described herein to a subject in need thereof.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1

Red bone marrow can be extracted or harvested from flat bones. Afterextraction, can selective lysis can be performed on the extracted bonemarrow or a desired cell population therein. This can be achieved byexposing the bone marrow to a lysing agent that selectively lyses redblood cells and white blood cells from the bone marrow but does not lysethe stem cells. One method of selectively lysing cells is to subject thecells to osmotic lysis. Osmotic lysis can be performed using water orweak acetic acid (less than or equal to 0.5% v/v). The lysing agent maybe heated prior to exposure to the bone marrow cells. An anticoagulantmay be added to the lysing agent to decrease platelet activation. Theimplant can then be treated with antibiotics and/or antifungals tofurther decontaminate the implant. After antibiotic treatment, theimplant may be combined with demineralized bone and placed into acryoprotectant prior to freezing. Freezing may be done in a stepwisemanner to more slowly freeze the stem cells and preserve cell viabilityand function.

Example 2

An enriched stem cell implant may be produced by marrow rich bone wherethe bone marrow cells are not extracted from the bone but rather beincluded with and/or attached to cancellous bone. A rinsing agent, suchas saline, can be used to wash away unattached cells which eliminatesthe majority of red blood cells and white blood cells. The rinsing agentmay be heated prior to exposure to the bone marrow cells. Ananticoagulant may be added to the rinsing agent to decrease plateletactivation. The implant may then be treated with antibiotics and/orantifungals to further decontaminate the implant. After antibiotictreatment, the implant may be combined with demineralized bone andplaced into a cryoprotectant prior to freezing. Freezing may be done ina stepwise manner to more slowly freeze the stem cells and preserve cellviability and function.

Example 3

An enriched stem cell implant that can contain viable bone marrow stemcells can be produced by utilizing bone marrow rich bone where bonemarrow cells are included with and/or attached to cancellous bone, suchas granules, blocks, or structures. A lysing agent, such as water or0.5% (v/v) acetic acid, can be used to wash away and lyse unattachedcells which eliminates the majority of red blood cells and white bloodcells. The lysing agent can also selectively lyse white blood cellsattached to the bone such as granulocytes. The lysing agent can beheated prior to exposure to the bone marrow cells. An anticoagulant canbe added to the lysing agent to decrease platelet activation. Theimplant can then be treated with antibiotics and/or antifungals tofurther decontaminate the implant. After antibiotic treatment, theimplant can be combined with demineralized bone and placed into acryoprotectant prior to freezing. Freezing can be done in a stepwisemanner to more slowly freeze the stem cells and preserve cell viabilityand function.

Example 4

The implant containing bone marrow stem cells may be tested forsterility after production by determining bioburden or by methods of USP71. The implant can be stored at temperatures around −80° C. or colderand shipped for implantation on dry ice or liquid nitrogen. Afterproduction of the implant, cell viability can be evaluated by soakingthe implant in collagenase to release the attached stem cells andobserved under a microscope with trypan blue staining. Stem cellfunction and osteogenic differentiation ability can also be determinedby culturing the unattached stem cells in osteogenic medium. Prior toimplantation, the implant can be thawed and rinsed to remove anyremaining cryoprotectant.

We claim:
 1. A bone marrow stem cell composition or implant comprising:viable bone marrow stem cells, wherein the bone marrow stem cellcompositions is produced by the method comprising: harvesting bonemarrow from a donor; selectively lysing the bone marrow to obtain bonemarrow stem cells, wherein the step of selectively lysing is performedusing water or a solution containing 0.5% or less v/v acetic acid. 2.The bone marrow stem cell composition or implant of claim 1, wherein themethod of producing the bone marrow stem cell composition or implantfurther comprises the step of washing the harvested bone marrow prior toselectively lysing the bone marrow.
 3. The bone marrow stem cellcomposition or implant of claim 1, wherein the method of producing thebone marrow stem cell composition or implant further comprises the stepof centrifuging after the step of selectively lysing the bone marrow. 4.The bone marrow stem cell composition or implant of claim 1, wherein themethod of producing the bone marrow stem cell composition or implantfurther comprises the step of filtering after the step of selectivelylysing the bone marrow.
 5. The bone marrow stem cell composition orimplant of claim 1, wherein the method of producing the bone marrow stemcell composition or implant further comprises the step of adding astabilization solution after the step of selectively lyisng the bonemarrow.
 6. The bone marrow stem cell composition or implant of claim 1,wherein the composition or implant further comprises a scaffoldmaterial.
 7. The bone marrow stem cell composition or implant of claim6, wherein the scaffold material is allograft bone.
 8. The bone marrowstem cell composition or implant of claim 6, wherein the scaffoldmaterial is allograft demineralized bone
 9. A method of making an bonemarrow stem cells composition or implant containing viable stem cells,the method comprising: harvesting bone marrow from a donor; selectivelylysing the bone marrow to obtain bone marrow stem cells, wherein thestep of selectively lysing is performed using water or a solutioncontaining 0.5% or less v/v acetic acid.
 10. The method of claim 9,wherein the method further comprises the step of washing the harvestedbone marrow prior to selectively lysing the bone marrow.
 11. The methodof claim 9, wherein the method further comprises the step ofcentrifuging after the step of selectively lysing the bone marrow. 12.The method of claim 9, wherein the method further comprises the step offiltering after the step of selectively lysing the bone marrow.
 13. Themethod of claim 9, wherein the method further comprises the step ofadding a stabilization solution after the step of selectively lyisng thebone marrow.
 14. The method of claim 9, further comprising the step ofmixing the bone marrow stem cell composition or implant with a scaffoldmaterial after the step of selectively lysing the bone marrow.
 15. Themethod of claim 14, wherein the scaffold material is allograft bone. 16.The method of claim 9, further comprising the step of adding a scaffoldmaterial after the step of harvesting the bone marrow.
 17. The method ofclaim 16, wherein the scaffold material is allograft bone.
 18. A methodof treating a subject in need thereof, the method comprising:administering an amount of a bone marrow stem cell composition orimplant to a subject in need thereof wherein the bone marrow stem cellcomposition or implant is as according to claim
 1. 19. The method ofclaim 18, wherein the bone marrow stem cell composition or implantfurther comprises a scaffold and wherein the viable bone marrow stemcells are attached to or otherwise associated with the scaffold.
 20. Themethod of claim 19, wherein the scaffold is allograft bone.